面肩肱型肌营养不良症DUX4基因表达的调控机制研究

Facioscapulohumeral muscular dystrophy (FSHD) is the most common adult form of muscular dystrophy (MD), with a prevalence of approximately 1:20,000. It is an autosomal dominant disorder. The spectrum of disease severity varies widely with high disability. It results from deletion of a critical number of a repetitive 3.3-kb microsatellite sequence known as D4Z4 repeats on chromosome 4. Additionally, D4Z4 reduction is pathogenic only in a few genetic backgrounds including a special sequence of SSLP proximal to the D4Z4 repeat and the 4qA polymorphism distal to the repeat. The molecular genetic basis of FSHD is so complex that the disease mechanism has not been entirely elucidated. The inefficient repeat-mediated epigenetic repression of D4Z4 repeats is supposed to cause the variegated expression of DUX4 retrogene, encoding a double-homebox transcription factor, in skeletal muscle and result in a deregulation cascade of apoptosis, impaired muscle regeneration, and induction of an immune response. Since some disagreements regarding the details of mechanisms remain in the field, efforts are largely focusing on the dominant pathway and pathophysiological consequences of the DUX4 dysregulation. Our previous study in a large cohort of patients has confirmed that the specific sequence haplotype of SSLP161-D4Z4/PAS-4qA is almost uniquely associated with Chinese FSHD, which supports the prominent role for DUX4 gene and its polyadenylation sequence. Based on these evidences, we will utilize the new gene edit technology for multi-targeted knockout in vitro to accurately regulate the expression of DUX4 gene, which aim to identify the plausible mechanism of DUX4 regulation and explore the function of the flanking sequences of the D4Z4 repeats. Furthermore, we are going to apply BAC vector system to generate transgenic mouse model carrying large inserted DNA segments harboring the permissive haplotype SSLP161-D4Z4/PAS-4qA, which will provide a generally accepted and coherent FSHD animal model more similarly to the human genetic background. Then we are able to investigate the mechanism of DUX4 regulation in vivo and to yield new avenues for therapeutic development.

面肩肱型肌营养不良症（FSHD）是最常见的成人肌营养不良症，致残率高，临床异质性大。基因定位于4q35亚端粒区，与一段大卫星序列（D4Z4 repeats）的多拷贝缺失直接相关，其上下游特异性序列SSLP和4qA/4qB具有致病选择性，但发病机制尚未阐明，推测D4Z4拷贝数减少导致内部DUX4基因异常表达，引起肌肉损害效应，但DUX4基因的具体调控途径及作用机制存在诸多矛盾，是研究的核心问题。我们前期证实中国人FSHD的致病基因型仅为SSLP161-D4Z4/PAS-4qA，支持DUX4基因的关键作用，本项目以此核心结构为切入点，采用新型基因编辑技术在细胞水平实现多靶点精准基因敲除，体外调控DUX4基因的表达，以期证实DUX4基因及侧翼序列的功能，通过BAC载体系统构建大片段插入的转基因鼠模型进行功能验证，体内调控DUX4基因表达，探索FSHD的发病机制，寻找潜在治疗靶点。

面肩肱型肌营养不良症（FSHD）是最常见的成人型肌营养不良症，致残率较高，发病机制高度特殊且复杂，是一种新型的表观遗传效应（Epigenetic etiology）模式疾病。本项目以临床导向结合基础研究，多途径多层次分析表观遗传效应在表型异质性中的关键作用。首先通过国际注册登记和平台化管理，建立标准化临床队列，进行基因型-表型及其与4q35-DRs区域特异性甲基化改变的相关性研究，证实FSHD1患者甲基化显著降低，与发病年龄呈正相关，而与临床严重程度呈负相关；家系内表型严重者的甲基化水平显著下降，而表型外显不全或未外显者，甲基化水平显著高于典型患者。对亚队列的临床、分子遗传学特征及其表观作用做了总结，尤其是发现嵌合体型FSHD携带的致病D4Z4重复相对短，嵌合比例相对高，提示突变发生在有丝分裂的早期阶段，而标准嵌合染色体型占97%，提示有丝分裂期间不携带交叉互换（crossover）的染色体交换（interchromosomal gene conversion）是嵌合体型形成的主要机制。首次发现SMCHD1基因起始密码子杂合突变（c.1 A>G）导致中国FSHD2，证实FSHD2与FSHD1存在共同基因结构——SSLP161-D4Z4/PAS-4qA，提示两型有共同的表观调控通路。采用三代测序Nanopore结合Bionano技术进行FSHD全基因精细测序、甲基化谱绘制及单倍体组装，形成中国人FSHD的共识序列，对后续微结构变异、转座子及基因修复提供结构基础。将FSHD患者外周血白细胞重编程为iPSCs，作为后续蛋白功能及修复研究的细胞工具；引进FLExDUX4及STOCK Tg (ACTA1-cre/Esr1*)2Kesr/J（ACTA1-MCM）小鼠模型，构建cre-dux4双转基因模型鼠，进行了肌肉病理学和行为学等研究，用于后续体内靶向DUX4甲基化修饰及治疗的探索。